PROJECT SUMMARY Nearly 50% of women over the age of 50 experience recurrent urinary tract infections. Additionally, over 25% of older women experience burdensome lower urinary tract symptoms including urinary urgency, incontinence, and painful urination. In the US alone, over $2 billion are spent each year to treat these disorders. With 566 million people ?65 years old worldwide now and estimates of nearly 1.5 billion by 2050, it is crucial that we determine the pathophysiologic basis of these disorders so as to treat and prevent them. This proposal will test the central hypothesis that aging leads to microbial dysbiosis and elevation of pro-inflammatory signals in the bladder mucosa, which cause formation of organized bladder tertiary lymphoid follicles (BTLFs). This hypothesis is founded on solid preliminary evidence that aggregates in both mouse and human bladders have features of tertiary lymphoid follicles, which result from diverse chronic inflammatory mechanisms in other mucosal tissues such as the gut and lung. Aim 1, to define the cellular composition, activation status, and functional capacity of BTLFs in aged mice, will test the hypothesis that BTLFs act as sites of antigen presentation and antibody production. Aim 2, to define the inflammatory mechanisms driving bladder BTLF formation, will test the hypothesis that BTLF formation requires both classical lymphotoxin signaling by LT?1?2 and inflammatory mediators such as TNF?, whose expression are increased during aging. Aim 3, to identify microbial stimuli that drive BTLF formation, will test the hypothesis that BTLF formation in mice is stimulated by chronic inflammation in response to microbial exposure during recurrent urinary tract infections or age- associated microbiota alterations. Completion of these aims will provide insight into the mechanisms, mediators, and microbial triggers governing the development, function, and maintenance of BTLFs. By doing so, this work will provide important insights regarding the mucosal immunity of the bladder, especially in chronic inflammation and response to recurrent urinary tract infections in the elderly. Additionally, this work will establish a platform to develop and identify biomarkers of age-related immune function changes that can be used to guide future clinical trials and treatment strategies to improve responses to infections in the elderly.